ELISA assay (acronym of the term “Enzyme-Linked ImmunoSorbent Assay”) is commonly used for quantitative diagnosis of molecular markers (antigens or antibodies) which are present in fluids, biopsies, cultures, or any other sample.
This assay is used more particularly for diagnosing infections (HIV, HPV, etc.), the condition of the cardiovascular and immune system (Troponin C, specific antibodies, etc.), and the presence and development of cancers (PSA, CEA, etc.).
This technique, which is presently the most robust and most widespread of methods of diagnosis, is nevertheless not without drawbacks: specifically, its complexity, the use of expensive automated instruments, and its duration, which may extend to several hours.
ELISA assay is a heterogeneous-phase immunoassay technique, meaning that it requires a solid support (typically, a titration plate comprising a plurality of wells) to which a molecule is attached beforehand, said molecule being adapted for capturing the molecule to be assayed.
When the molecule of interest has been captured on said support, washing allows the remainder of the sample to be removed and the step of detecting and quantifying said molecule to be commenced.
For example, in the case of the “sandwich” ELISA assay, for assaying an antigen in a solution, the surface of the support is covered with a specified amount of a capture antibody, said antibody being adapted for binding to the target antigen.
The solution likely to contain said antigen is then applied to the support; said antigen then binds to the capture antibody sited on the surface of the support.
The support is subsequently washed so as to remove the unbound antigen possibly remaining in the solution.
Deposited next on the support is a solution containing an antibody called a detection antibody, which is adapted for binding to the antigen fixed on the support, and which, moreover, is coupled to an enzyme.
A further washing step is implemented, so as to retain the antigen bound to the detection antibody on the support, said antibody being itself coupled to the enzyme.
Lastly, for the detection and quantification of the antigen, a substrate is deposited on the support, this substrate being converted by the enzyme into a detectable signal (for example, a color, or spectroscopically, in other words by emission of fluorescence) that is representative of the binding between the antigen and the detection antibody.
Said signal may be observed with the naked eye or by means of an instrument, such as a spectrophotometer.
ELISA assay encompasses various implementations, but they all include multiple steps of grafting and incubation which are separated by washing steps.
The substantial duration of ELISA assay is due essentially to the succession of the various steps.
Variants of this assay which employ magnetic microbeads have already been described.
The article by D. Issadore et al., Self-assembled magnetic filter for highly efficient immunomagnetic separation, Lab Chip, 2011, 11, 147 thus describes a method for capturing a molecule in a sample by circulating said sample within a fluid microchannel arranged below a polydimethylsiloxane (PDMS) matrix in which magnetic grains of NdFeB have been immobilized.
Owing to the method of manufacture employed for said support, the grains are distributed randomly within the thickness of the matrix, and their magnetic orientation is random.
Each grain generates a magnetic field gradient which is capable of attracting magnetic particles with a diameter of 1 μm that are coupled to the molecules to be detected, to form complexes.
However, the smaller the size of the particles to be captured, the less effective a magnetic field gradient of this kind, meaning that this device performs less well for the capture of magnetic particles with sizes smaller than 1 μm, of the order of 500 nm or less, for example, and especially of nanoparticles.
Furthermore, the capture of the magnetic particles is relatively slow.
This capturing step is followed by washing of the surface of the PDMS matrix and then by fluorescence detection of the complexes immobilized on said surface.